Isothiocyanato-azobenzene-sulfonic, arsonic and substituted-glutamic acid haptens

ABSTRACT

Compounds of formula (I) ##STR1## in which n is 1 or 2, R 1  is C 1-4  alkyl, C 1-4  alkoxy, nitro, amino, halo or hydroxy, m is 0, 1 or 2, R 2  is an immunogenic determinant and x is 1 or 2, provided that when n is 1, x is 1 or 2 and there is one diazo moiety at the 3-position or two diazo moieties at the 3- and 5-positions, with respect to the isothiocyanate group, and the hydroxy group is at the 4-position; and provided that when n is 2, x is 1 and the diazo moiety is at the 4-position and the hydroxy groups are at the 3- and 5-positions, are described. The compounds are useful in the preparation of immunoglobulin conjugates for employment in diagnostic techniques.

This invention relates to novel compounds and their use in thepreparation of novel hapten-immunoglobulin conjugates.

Techniques of studying antigen distribution in cell and tissue sectionpreparations for the purpose of diagnosis are well known. Antibodiessuitably marked with, for example, fluorescent material bind to antigenon the cell surface which is thus conveniently labelled. In onetechnique by indirect labelling, a second-layer antibody coupled to asuitable marker is employed to label a first-layer antibody specific fora particular antigen. The first-layer antibody can be modified bycovalent linkage with one or more hapten molecules to form ahapten-antibody conjugate which in turn is detected by labelledantihapten antibody, see review by Wofsy L., Henry C. and Cammisuli S.in Contemp. Top. Mol. Immunol. 7.215. Such a labelling technique canalso be applied to other immunoglobulin materials which may find utilityin assay and control experiments.

In preparing the hapten-immunoglobulin conjugate it is, of course,important that the antibody or other immunoglobulin material, should notbe seriously damaged and attachment of hapten is best effected by meansof a reagent consisting of a hapten linked to a suitable coupling group.

The present invention provides a novel reagent which is convenientlyprepared and readily handled and which can be employed under mildconditions to prepare hapten-immunoglobulin conjugates withoutappreciable loss of utility. The reagent of the invention is a compoundof the formula (I) ##STR2## in which n is 1 or 2, R¹ is C₁₋₄ alkyl, C₁₋₄alkoxy, nitro, amino, halo or hydroxy, m is 0, 1 or 2, R² is animmunogenic determinant and x is 1 or 2; provided that when n is 1, x is1 or 2 and there is one diazo moiety at the 3-position or two diazomoieties at the 3- and 5-positions, with respect to the isothiocyanategroup, and the hydroxy group is at the 4-position; and provided thatwhen n is 2, x is 1 and the diazo moiety is at the 4-position and thehydroxy groups are at the 3- and 5-positions. The compounds of theinvention are thus mono or bis azo derivatives of 4-hydroxy- or3,5-dihydroxy-phenylisothiocyanate.

In the above formula (I), R² can be any suitable immunogenic determinantoptionally in its various optical or racemic forms, and for exampleincludes an arsonate group (--AsO₃ H₂), a sulphonate group (--SO₃ H), anitro group (--NO₂), a carboxyl group (--CO₂ H), a trimethylammoniumgroup (Me₃ N--) a group derived from glutamic acid ##STR3## or a groupderived from glycine [--CONHCH₂ CO₂ H] the preferred groups beingarsonate, sulphonate and the groups derived from glutamic acid andglycine. The group R¹ when it is C₁₋₄ alkyl can be straight or branchedchain and can be for example methyl, ethyl, propyl, isopropyl or butyland is preferably methyl. When R¹ is C₁₋₄ alkoxy it is a C₁₋₄ alkylgroup linked via an oxygen atom to the phenyl nucleus, and is preferablymethoxy. When R¹ is halo it can be chloro, bromo or iodo and ispreferably chloro. The most conveniently obtainable compounds are thosein which m is o and a preferred group of components is one of thefollowing formula: ##STR4##

The invention also includes a method of preparing a compound of formula(I) which comprises reacting a phenylisothiocyanate of formula ##STR5##in which n is 1 and the hydroxy group is at the 4-position or n is 2 andthe hydroxy groups are at the 3- and 5-positions, with a diazonium saltof formula ##STR6## in which R¹, m and R² are as defined for formula (I)and X.sup.⊖ is an anion.

Reactants of formulae (II) and (III) are either known compounds or canbe prepared from known compounds by simple chemical methods well knownin the art. For example, the starting materials of formula (III) areprepared from arsanilic acid, p-aminobenzoyl glutamic acid, sulphanilicacid or derivatives thereof, which are readily diazotised by wellrecognized methods, employing for example sodium nitrite and dilutehydrochloric acid, to give the appropriate diazonium salt. The anion X⊖is preferably a halid ion, for example chloride.

Compounds (II) and (III) are preferably reacted together in an aqueousmedium at a temperature of from 0° C. to 5° C., the pH being maintainedat from 8 to 10, for example at pH 9. If desired a water misciblesolvent such as dimethyl formamide or tetrahydrofuran can be added, andthe products of the process can be purified by recrystallisation or bygel filtration.

The compounds of the invention are convenient reagents that can beeasily prepared. They are solids which are readily purified and preparedin high yield. In these respects they represent an improvement overprior art materials. Moreover they can be reacted under mild conditionswith antibody to produce hapten-antibody conjugates with high yield andwithout significantly affecting the net charge or other physicalproperties of the antibody as to alter its binding activity.

The invention also provides a hapten-immunoglobulin conjugate which isan immunoglobulin modified with one or more, for example 5 to 20, groupsof the formula ##STR7## in which n, R¹, m, R² and x have the valuesdefined in formula (I); and a process for preparing ahapten-immunoglobulin conjugate which comprises reacting animmunoglobulin with a compound of formula (I). Preferably theimmunoglobulin is an antibody.

The antibodies are obtained by well known procedures from the serum ofimmunised animals or by culturing hybridomas secreting monoclonalproducts.

Antibodies can be haptenated according to this method under mildconditions of pH and temperature with chemical modification occurringsolely at free amino groups. For example the reaction can be effected ata temperature of from 10° C. to 25° C. such as at room temperature, in asuitable aqueous medium buffered to a pH of between 7.5 to 8.6. Theresulting conjugate can be purified by gel filtration and stored insaturated ammonium sulphate solution, being readily brought back intosolution by dialysis employing, for example, borate buffer or,alternatively, it can be stored in a refrigerator at 4° C. or frozen atfor example -20° C.

The reaction results in one or more molecules of formula (I) beingattached to the immunoglobulin and the number of molecules attached onthe conjugate depends on the concentrations of the reactants employedand the time for which the reaction is allowed to proceed. Evaluation ofthe conjugate can be carried out using techniques such as affinitychromatography, determination of cytotoxicity and labelling specificity.The hapten concentration in solutions of conjugates can be determined byspectroscopy at pH 13, making use of pre-determined molar extractioncoefficients at pH 13 of compounds of formula (I).

As has been indicated above, the hapten-antibody conjugates of theinvention are employed in conjunction with labelled antihaptenantibodies. These are easy to prepare and can be purified readily byaffinity chromatography (Cuatrecases and Anfinsen, Methods inEnzymology, 34, 345 et seq., Academic Press, New York)

This system of indirect labelling can be used to discriminate betweentwo antigens on the same cell or between two cells with distinguishingantigens. The procedure is especially suited to a situation in whicheach of two antigens is best visualized by the indirect method, sincethis can be done by the use of two noncrossreactive antihaptenantibodies and for example antibodies with different fluorochromes canbe used to distinguish clearly a variety of antigens on human and animalcell surfaces. This procedure is also suited to situations whereincreased sensitivity is required, by making use of the greateramplification achieved by using highly heptenised antibody.

Examples of preferred antibody for use in the immunoglobulin conjugatesof the invention include anti-human Ig, anti-human Ia, anti-human Leu-1,anti-human T-cell markers, and anti-mouse Thy 1,2.

The invention is illustrated by the following Examples.

EXAMPLE 1 Preparation of2-hydroxy-5-isothiocyanato-azo-benzene-4'-arsonic acid

Sodium nitrite (69 mg, 1 mmole) in water (1.5 ml) was added dropwise toa cooled (<5° C.) solution of arsanilic acid (217 mg. 1 mmole) in 1 NHCl (2.5 ml) and the mixture stirred at <5° C. for 30 minutes.

0.34M Borate buffer, pH 9.3(10 ml) was added to a solution ofp-hydroxyphenylisothiocyanate (151 mg, 1 mmole) in DMF (2 ml) and theresulting emulsion cooled to 5° C. The diazonium solution was addeddropwise with stirring at <5° C. over 5 minutes, the pH being maintainedat pH 9±0.2 by the addition of <5 N NaOH. The reaction mixture wasstirred at room temperature for 1 hour, then washed with ether (2×20 ml)and acidified to pH 1. The precipitated crude product was washed with 1N HCl (20 ml) and water (20 ml), and redissolved in 0.1 M borate bufferpH 8.6 (5 ml). This solution was chromatographed at 4° C. on a 1.5×112cm (200 ml) column of Bio-Gel P-2 (100-200 mesh) equilibrated with 0.1 Mborate buffer pH 8.6. Fractions (8 ml) were collected and assessed by600-400 nm scans aliquots diluted 1:30 with water. Fractions 17-24 werecombined and acidified (pH-1), and the precipitated product washed withwater (2×15 ml), suspended in water and freeze dried. λmax(0.1 N NaOH)505 nm (8900)(m.p.>260° C.)

The following compounds were similarly prepared2-Hydroxy-5-isothiocyanato-azo-benzene-4'-sulphonic acid (fromsulphanilic acid), λmax(0.1 N NaOH) 510 nm (ε9100)(m.p.>260° C.L-2-(2-hydroxy-5-isothiocyanato-azo-benzene-4'-carboxamido)-glutaricacid (from L-p-aminobenzoylglutamic acid), λmax(0.1 N NaOH) 510nm(ε10900). (m.p. 98° C.).

EXAMPLE 2 Preparation of L-benzoyl-glutamic acid-conjugated mouse IgG₂anti-human Ia

100 μl of a solution ofL-2-(2-hydroxy-5-isothiocyanato-azo-benzene-4'-carboxamido)-glutaricacid (4 mg/ml 0.34 M borate buffer pH 8.6 ) was added to a solution ofmouse IgG₂ anti-human Ia(2.8 mg) in the same buffer (0.4ml). The mixturewas stored in the dark at room temperature for 18 hours, thenchromatographed on a 1.0×16.5 cm (13 ml) column of Biogel P-6 (suppliedby Bio-Rad Laboratories Limited) equilibrated with buffer. The excludedpeak (2.4 ml) was collected, and contained 1.2 mg/ml of conjugate having8.2 moles L-benzoylglutamic acid/mole of antibody.

EXAMPLE 3 Preparation of arsanilic acid-conjugated mouse IgG₁anti-carcino-embryonic antigen

120 μof a solution of 2-hydroxy-5-isothiocyanato-azo-benzene-4'-arsonicacid (4 mg/ml 0.34 M borate buffer pH 8.6) was added to a solution ofmouse IgG₁ anti-carcinoembryonic antigen (4 mg) in the same buffer (0.5ml). The mixture was stirred in the dark at room temperature. After 6hours a 0.25 ml aliquot was chromatographed on a 1.0×15.5 cm (12 ml)column of Ultrogel AcA-54 (LKB Instruments Ltd) equilibrated withbuffer. The excluded peak (2.8 ml) was collected and contained 0.6 mg/mlof conjugate having 8.8 moles arsanilic acid/mole of antibody.

After 24 hours the remainder of the reaction mixture was similarlychromatographed. The excluded peak (3.4 ml) contained 0.6 mg/ml ofconjugate having 15.2 moles arsanilic acid/mole of antibody.

I claim:
 1. A compound of formula ##STR8## in which n is 1 or 2, R¹ isC₁₋₄ alkyl, C₁₋₄ alkoxy, nitro, amino, halo or hydroxy, m is 0, 1 or 2,R² is an immunogenic determinant and x is 1 or 2, provided that when nis 1, x is 1 or 2 and there is one diazo moiety at the 3-position or twodiazo moieties at the 3- and 5-positions, with respect to theisothiocyanate group, and the hydroxy group is at the 4-position; andprovided that when n is 2, x is 1 and the diazo moiety is at the4-position and the hydroxy groups are at the 3- and 5-positions, whereinthe term diazo moiety represents ##STR9##
 2. A compound according toclaim 1 in which the immunogenic determinant is an arsonate (--A_(s) O₃H₂) group, a sulphonate group (--SO₃ H) or ##STR10##
 3. A compoundaccording to either of claims 1 or 2 in which n is 1, x is 1 and m is o.